Gene expression using T cell factor responsive element

ABSTRACT

Disclosed are DNA elements and constructs useful for obtaining tumour-selective gene expression in tumours having a mutated β-catenin/APC pathway. In particular, the use of these constructs to express genes encoding therapeutic proteins in colorectal cancer cells is described. The constructs comprise multiple repeats of a TCF-binding element operably linked to a promoter. By means of such a construct, tumour cell-specific expression of a prodrug-converting enzyme such as nitroreductase may be achieved. Coupled with systemic administration of a suitable prodrug, such as CB1954, selective killing of such tumour cells can be demonstrated.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation application which claimspriority under 35 U.S.C. § 119(a)-(d) of United Kingdom Application No.0005099.7, filed Mar. 2, 2000 and also claims the benefit under 35U.S.C. § 119(e) of U.S. Application Serial No. 60/187,465, filed Mar. 6,2000, and claims benefit under 35 U.S.C. § 119(a)-(d) of PCT ApplicationNo. PCT/GB01/00856, filed Mar. 1, 2001, all applications incorporatedherein by reference in their entireties.

BACKGROUND TO THE INVENTION

[0002] The present invention relates to a T cell factor (TCF)-responsiveelement, a gene and uses of the TCF-responsive element or nucleic acidconstruct in assays nucleic acid construct comprising a TCF-responsiveelement and a therapeutic and therapy.

[0003] TCFs are a family of transcription factors within the HighMobility Group (HMG) of DNA-binding proteins (Love et al., Nature, 376,791-795,1995). The family includes TCF-1, TCF-3 and TCF-4 which aredescribed in van der Wetering et al, (EMBO J., 10, 123-132, 1991),EP-A-0 939 122 and Korinek et al. (Science, 275, 1784-1787, 1997). TCF-4has been shown to be involved in tumorigenesis related to Wnt/Winglesssignalling. TCF and LEF-1 (lymphoid enhancer factor-1) are considered tomediate a nuclear response to Wnt signals by interacting with β-catenin.Wnt signalling and other cellular events that increase the stability ofβ-catenin are considered to result in transcriptional activation ofgenes by LEF-1 and TCF proteins in association with β-catenin. In theabsence of Wnt signalling, LEF-1/TCF proteins repress transcription inassociation with Groucho and CBP (CREB binding protein).

[0004] In the absence of Wnt signalling, β-catenin is found in twodistinct multiprotein complexes. One complex, located at the plasmamembrane, couples cadherins (calcium dependent adhesion molecules) withthe actin cytoskeleton whereas the other complex (containing theproteins adenomatous polyposis coli protein (APC), axin and glycogensynthase kinase 3β (GSK3β)) targets β-catenin for degradation. Wntsignalling antagonises the APC-axin-GSK3β complex, resulting in anincrease in the pool of free cytoplasmic β-catenin. The free cytoplasmicβ-catenin can translocate to the nucleus where it binds LEF-1/TCFfactors and activates Wnt target genes. The regulation of LEF-1/TCFtranscription factors by Wnt and other signals is discussed in Eastmanet al, (Current Opin. Cell Biology, 11, 233-240,1999).

[0005] The APC gene is a tumour supressor gene that is inactivated inmost colorectal cancers. Mutations of APC are considered to cause theaccumulation of free β-catenin, which then binds TCF causing increasedtranscriptional activation of genes including genes important for cellproliferation (e.g. cyclin D1 (Tetsu et al., Nature 398, 422-426, 1999and Shtutman et al., PNAS USA, 96, 5522-5527, 1999) and c-myc (He etal., Science, 281, 1509-1512,1998)). The involvement of APC in tumourdevelopment is discussed in He et al, (supra).

[0006] TCFs are known to recognise and bind TCF binding elements whichhave the nucleotide sequence CTTTGNN, wherein N indicates A or T (vander Wetering et al, supra).

[0007] TCF reporter genes have been constructed and are described inKorinek et al, (Science, 275, 1784-1787,1997), Morin et al, (Science,275,1787-1790, 1997), EP-A-0 939 122 and WO 98/41631. The TCF reportergene is said to comprise three TCF binding elements upstream of either aminimal c-Fos promoter driving luciferase expression or a minimal herpesvirus thymidine kinase promoter driving chloramphenicolacetyl-transferase expression. He et al (supra) discloses TCF reportergene constructs comprising four TCF binding elements inserted intopBV-Luc.

[0008] There is a need for an effective treatment of cancers associatedwith a deregulation of the Wnt signalling pathway. Such cancers includemost colorectal cancers, approximately 30% of melanomas and some breast,prostate and hepatocellular carcinomas.

[0009] There is also a need for a TCF response element which when linkedto an expressible gene gives improved levels of expression andspecificity.

SUMMARY OF THE INVENTION

[0010] The present invention provides a nucleic acid constructcomprising:

[0011] a TCF response element comprising:

[0012] at least one TCF binding element having the sequence CTTTGNN,wherein N is A or T; and

[0013] a promoter,

[0014] and an expressible therapeutic gene operably linked to the TCFresponse element,

[0015] wherein the TCF response element enables inducible expression ofthe operably linked therapeutic gene.

[0016] The term “inducible expression” as used herein means the level ofexpression obtained using the TCF response element is induced (i.e.increased) when one or more TCF/β catenin heterodimers binds to one ormore of the TCF binding elements. Preferably the level of expression isincreased by at least 15 fold, more preferably at least 25 fold and mostpreferably at least 30 fold.

[0017] The term “operably linked” as used herein refers to a cis-linkagein which the gene is subject to expression under control of the TCFresponse element.

[0018] The expressible gene comprises the necessary elements enablinggene expression when operably linked to the TCF response element, suchas splice acceptor sequences, internal ribosome entry site sequences(IRES) and transcription stop sites. Such elements are well known tothose skilled in the art.

[0019] It has been found that by using the nucleic acid construct of thepresent invention that expression of the operably linked therapeuticgene is only induced when TCF/β catenin heterodimers are present andcapable of activating transcription. As cells that have become cancerousdue to the deregulation of the Wnt signalling pathway have TCF/β cateninheterodimers, which activate transcription, expression of thetherapeutic gene will be induced. Accordingly, the nucleic acidconstruct of the present invention acts as a tumour selective promoter.

[0020] The nucleic acid construct of the present invention exhibitshighly selective expression in that it gives no induction of expressionof an operably linked gene above the background level in the absence ofTCF/β catenin heterodimers or a functionally equivalent transcriptionactivating factor.

[0021] The therapeutic gene can be any gene that on expression gives atherapeutic benefit. Preferred therapeutic genes include genes encodingtoxins such as ricin and diphtheria toxin, and prodrug activatingenzymes such as nitroreductases that activate CB1954, cytosine deaminasewhich activates 5-fluorocytosine, cytochrome P-450 which activatescyclophosphamide and paracetamol, and thymidine kinase which activatesganciclovir. Preferably the therapeutic gene encodes a nitroreductase.Suitable nitroreductases are described in EP-A-0638123 and Watanabe etal, (NAR, 18, 1059,1990). Other preferred therapeutic gene include genesencoding immunomodulatory agents such as IL-2, IL-12, GMCSF, B7-1 andB7-2 co-stimulatory molecules; genes encoding tumour suppressers such asRB, p53 and p16; and genes encoding apoptotic genes such as Bax, FasLand caspases.

[0022] The promoter can be any promoter that gives a desired level ofexpression of the operably linked gene. Suitable promoters include theSV40 promoter, the E1B promoter, and the c-Fos promoter. Preferably thepromoter is the basal TATA box of the E1B promoter.

[0023] Preferably the TCF response element contains at least three andmore preferably at least five TCF binding elements. It has been foundthat by using at least three and more preferably at least five TCFbinding elements that an unexpected increase in expression can beobtained compared to a TCF response element containing fewer bindingelements. This increase in expression is desirable for the production ofa therapeutically effective amount of an encoded product.

[0024] Preferably the TCF response element comprises between 5 and 15TCF binding elements, more preferably between 5 and 10 TCF bindingelements and most preferably 5 TCF binding elements.

[0025] The TCF binding elements are preferably separated from each otherby between 3 and 20 nucleotides, more preferably by between 3 and 14 andmost preferably by between 10 and 12 nucleotides.

[0026] It is further preferred that the TCF binding elements are sospaced from each other as to be equally distributed radially around theDNA helix, especially when the promoter is the E1B promoter.

[0027] It is preferred that the TCF binding element closest to thepromoter is between 140 and 10 nucleotides from the TATA box of thepromoter. It is further preferred that the TCF binding element closestto the promoter is between 100 and 10 nucleotides, more preferablybetween 50 and 10 and most preferably between 30 and 15 nucleotides fromthe TATA box of the promoter.

[0028] In one preferred embodiment the TCF binding elements areseparated from each other by between 3 or 4 nucleotides and the TCFbinding element closest to the promoter is 25 nucleotides from the TATAbox of the promoter.

[0029] The TCF binding elements preferably have the nucleotide sequenceCTTTGAT.

[0030] The TCF binding elements can be in either orientation withrespect to the promoter, namely 5′ to 3′ or 3′ to 5′.

[0031] The present invention also provides a nucleic acid constructdesignated herein as 5merTCF-E1BTATA, which is shown schematically inFIG. 6 and described in the materials and method section below.

[0032] The present invention also provides a TCF response elementcomprising:

[0033] at least five TCF binding elements; and

[0034] a promoter sequence,

[0035] wherein the TCF response element when operably linked to anexpressible gene gives inducible expression of the operably linked gene.

[0036] The TCF response element comprising at least five TCF bindingelements can be used to obtain inducible expression of any operablylinked gene such as a reporter gene or a therapeutic gene. Suitablereporter genes include luciferase, β-galactosidase and chloramphenicolacetyl transferase.

[0037] The TCF response element comprising at least five TCF bindingelements has been found to give improved (i.e. increased) levels ofexpression of an operably linked gene compared to a TCF response elementcomprising less than 5 TCF binding elements.

[0038] The TCF binding elements and the promoter of the TCF responseelement comprising at least 5 TCF binding elements are as defined above.

[0039] The present invention also provides a TCF reporter constructcomprising the TCF response element having at least 5 TCF bindingelements operably linked to a reporter gene.

[0040] The present invention also provides the use of the TCF reporterconstruct of the present invention in a method of identifying candidatedrugs for use in the treatment of cancers associated with thederegulation of the Wnt signalling pathway comprising the steps of:

[0041] contacting the TCF reporter construct with a test compound underconditions in which the reporter gene is transcribed; and

[0042] measuring the transcription of the reporter gene;

[0043] wherein a test compound which inhibits transcription of thereporter gene is a candidate drug for cancer treatment.

[0044] Preferably the step of contacting the TCF reporter construct isperformed in the presence of a lysate from a cell with a deregulated Wntsignalling pathway.

[0045] The present invention also provides a vector comprising thenucleic acid construct of the present invention or the TCF responsiveelement having at least five TCF binding elements of the presentinvention operably linked to an expressible gene.

[0046] The vector may be any vector capable of transferring DNA to acell. Preferably, the vector is an integrating vector or an episomalvector.

[0047] Preferred integrating vectors include recombinant retroviralvectors. A recombinant retroviral vector will include DNA of at least aportion of a retroviral genome which portion is capable of infecting thetarget cells. The term “infection” is used to mean the process by whicha virus transfers genetic material to its host or target cell.Preferably, the retrovirus used in the construction of a vector of theinvention is also rendered replication-defective to remove the effect ofviral replication of the target cells. In such cases, thereplication-defective viral genome can be packaged by a helper virus inaccordance with conventional techniques. Generally, any retrovirusmeeting the above criteria of infectiousness and capability offunctional gene transfer can be employed in the practice of theinvention.

[0048] Suitable retroviral vectors include but are not limited to pLJ,pZip, pWe and pEM, well known to those of skill in the art. Suitablepackaging virus lines for replication-defective retroviruses include,for example, ΦCrip, ΦCre, Φ2 and ΦAm.

[0049] Other vectors useful in the present invention include adenovirus,adeno-associated virus, SV40 virus, vaccinia virus, HSV and poxvirusvectors. A preferred vector is the adenovirus. Adenovirus vectors arewell known to those skilled in the art and have been used to delivergenes to numerous cell types, including airway epithelium, skeletalmuscle, liver, brain and skin (Hitt, M M, Addison C L and Graham, F L(1997) Human adenovirus vectors for gene transfer into mammalian cells.Advances in Pharmacology, 40: 137-206; and Anderson W F (1998) Humangene therapy. Nature, 392: (6679 Suppl): 25-30).

[0050] A further preferred vector is the adeno-associated (AAV) vector.AAV vectors are well known to those skilled in the art and have beenused to stably transduce human T-lymphocytes, fibroblasts, nasal polyp,skeletal muscle, brain, erythroid and haematopoietic stem cells for genetherapy applications (Philip et al., 1994, Mol. Cell. Biol., 14,2411-2418; Russell et al, 1994, PNAS USA, 91, 8915-8919; Flotte et al.,1993, PNAS USA, 90, 10613-10617; Walsh et al., 1994, PNAS USA, 89,7257-7261; Miller et al., 1994, PNAS USA, 91, 10183-10187; Emerson,1996, Blood, 87, 3082-3088). International Patent Application WO91/18088 describes specific MV based vectors.

[0051] Preferred episomal vectors include transient non-replicatingepisomal vectors and self-replicating episomal vectors with functionsderived from viral origins of replication such as those from EBV, humanpapovavirus (BK) and BPV-1. Such integrating and episomal vectors arewell known to those skilled in the art and are fully described in thebody of literature well known to those skilled in the art. Inparticular, suitable episomal vectors are described in WO98/07876.

[0052] Mammalian artificial chromosomes can also be used as vectors inthe present invention. The use of mammalian artificial chromosomes isdiscussed by Calos (1996, TIG, 12, 463-466).

[0053] In a preferred embodiment, the vector of the present invention isa plasmid. The plasmid may be is a non-replicating, non-integratingplasmid.

[0054] The term “plasmid” as used herein refers to any nucleic acidencoding an expressible gene and includes linear or circular nucleicacids and double or single stranded nucleic acids. The nucleic acid canbe DNA or RNA and may comprise modified nucleotides or ribonucleotides,and may be chemically modified by such means as methylation or theinclusion of protecting groups or cap- or tail structures.

[0055] A non-replicating, non-integrating plasmid is a nucleic acidwhich when transfected into a host cell does not replicate and does notspecifically integrate into the host cell's genome (i.e. does notintegrate at high frequencies and does not integrate at specific sites).

[0056] Replicating plasmids can be identified using standard assaysincluding the standard replication assay of Ustav et al., EMBO J., 10,449-457, 1991.

[0057] The present invention also provides a host cell transfected withthe vector of the present invention. The host cell may be any mammaliancell. Preferably the host cell is a rodent or mammalian cell.

[0058] Numerous techniques are known and are useful according to theinvention for delivering the vectors described herein to cells,including the use of nucleic acid condensing agents, electroporation,complexing with asbestos, polybrene, DEAE cellulose, Dextran, liposomes,cationic liposomes, lipopolyamines, polyornithine, particle bombardmentand direct microinjection (reviewed by Kucherlapati and Skoultchi, Crit.Rev. Biochem. 16:349-379 (1984); Keown et al., Methods Enzymol. 185:527(1990)).

[0059] A vector of the invention may be delivered to a host cellnon-specifically or specifically (i.e., to a designated subset of hostcells) via a viral or non-viral means of delivery. Preferred deliverymethods of viral origin include viral particle-producing packaging celllines as transfection recipients for the vector of the present inventioninto which viral packaging signals have been engineered, such as thoseof adenovirus, herpes viruses and papovaviruses. Preferred non-viralbased gene delivery means and methods may also be used in the inventionand include direct naked nucleic acid injection, nucleic acid condensingpeptides and non-peptides, cationic liposomes and encapsulation inliposomes.

[0060] The direct delivery of vector into tissue has been described andsome short-term gene expression has been achieved. Direct delivery ofvector into muscle (Wolff et al., Science, 247, 1465-1468,1990) thyroid(Sykes et al., Human Gene Ther., 5, 837-844, 1994) melanoma (Vile etal., Cancer Res., 53, 962-967, 1993), skin (Hengge et al., Nature Genet,10, 161-166,1995), liver (Hickman et al., Human Gene Therapy,5,1477-1483,1994) and after exposure of airway epithelium (Meyer et al.,Gene Therapy, 2, 450-460,1995) is clearly described in the prior art.

[0061] Various peptides derived from the amino acid sequences of viralenvelope proteins have been used in gene transfer when co-administeredwith polylysine DNA complexes (Plank et al., J. Biol. Chem.269:12918-12924 (1994)); Trubetskoy et al., Bioconjugate Chem. 3:323-327(1992); WO 91/17773; WO 92/19287; and Mack et al., Am. J. Med. Sci.307:138-143 (1994)) suggest that co-condensation of polylysineconjugates with cationic lipids can lead to improvement in gene transferefficiency. International Patent Application WO 95/02698 discloses theuse of viral components to attempt to increase the efficiency ofcationic lipid gene transfer.

[0062] Nucleic acid condensing agents useful in the invention includespermine, spermine derivatives, histones, cationic peptides, cationicnon-peptides such as polyethyleneimine (PEI) and polylysine. ‘Sperminederivatives’ refers to analogues and derivatives of spermine and includecompounds as set forth in International Patent Application WO 93/18759(published Sep. 30, 1993).

[0063] Disulphide bonds have been used to link the peptidic componentsof a delivery vehicle (Cotten et al., Meth. Enzymol. 217:618-644(1992)); see also, Trubetskoy et al. (supra).

[0064] Delivery vehicles for delivery of DNA constructs to cells areknown in the art and include DNA/poly-cation complexes which arespecific for a cell surface receptor, as described in, for example, Wuand Wu, J. Biol. Chem. 263:14621 (1988); Wilson et al., J. Biol. Chem.267:963-967 (1992); and U.S. Pat. No. 5,166,320).

[0065] Delivery of a vector according to the invention is contemplatedusing nucleic acid condensing peptides. Nucleic acid condensingpeptides, which are particularly useful for condensing the vector anddelivering the vector to a cell, are described in International PatentApplication WO 96/41606. Functional groups may be bound to peptidesuseful for delivery of a vector according to the invention, as describedin WO 96/41606. These functional groups may include a ligand thattargets a specific cell-type such as a monoclonal antibody, insulin,transferrin, asialoglycoprotein, or a sugar. The ligand thus may targetcells in a non-specific manner or in a specific manner that isrestricted with respect to cell type.

[0066] The functional groups also may comprise a lipid, such aspalmitoyl, oleyl, or stearoyl; a neutral hydrophilic polymer such aspolyethylene glycol (PEG), or polyvinylpyrrolidine (PVP); a fusogenicpeptide such as the HA peptide of influenza virus; or a recombinase oran integrase. The functional group also may comprise an intracellulartrafficking protein such as a nuclear localisation sequence (NLS), anendosome escape signal such as a membrane disruptive peptide, or asignal directing a protein directly to the cytoplasm.

[0067] The present invention also provides the nucleic acid construct,vector or host cell of the present invention for use in therapy.

[0068] Preferably, the nucleic acid construct, vector or host cell isused in the treatment of cancer.

[0069] The present invention also provides the use of the nucleic acidconstruct, vector or host cell of the present invention in themanufacture of a composition for use in the treatment of cancer.

[0070] The present invention also provides a method of treatment,comprising administering to a patient in need of such treatment aneffective dose of the nucleic acid construct, vector or host cell of thepresent invention. Preferably, the patient is suffering from cancer.

[0071] Preferably, the cancer is any cancer associated with thederegulation of the Wnt signalling pathway such as colorectal cancer,melanomas, breast, prostate and hepatocellular carcinomas.

[0072] The present invention also provides a pharmaceutical compositioncomprising the nucleic acid construct, vector or host cell of thepresent invention in combination with a pharmaceutically acceptableexcipient.

[0073] The pharmaceutical compositions of the present invention maycomprise the nucleic acid construct, vector or host cell of the presentinvention, if desired, in admixture with a pharmaceutically acceptablecarrier or diluent, for therapy to treat a disease.

[0074] The nucleic acid construct, vector or host cell of the inventionor the pharmaceutical composition may be administered via a route whichincludes systemic, intramuscular, subcutaneous, intradermal,intravenous, aerosol, oral (solid or liquid form), topical, ocular, as asuppository, intraperitoneal and/or intrathecal and local directinjection.

[0075] The exact dosage regime will, of course, need to be determined byindividual clinicians for individual patients and this, in turn, will becontrolled by the exact nature of the protein expressed by thetherapeutic gene and the type of tissue that is being targeted fortreatment.

[0076] The dosage also will depend upon the disease indication and theroute of administration.

[0077] The amount of nucleic acid construct or vector delivered foreffective treatment according to the invention will preferably be in therange of between about 50 ng-1000 μg of vector DNA/kg body weight; andmore preferably in the range of between about 1-100 μg vector DNA/kg.

[0078] Although it is preferred according to the invention to administerthe nucleic acid construct, vector or host cell to a mammal for in vivocell uptake, an ex vivo approach may be utilised whereby cells areremoved from an animal, transduced with the nucleic acid construct orvector, and then re-implanted into the animal. The liver, for example,can be accessed by an ex vivo approach by removing hepatocytes from ananimal, transducing the hepatocytes in vitro and re-implanting thetransduced hepatocytes into the animal (e.g., as described for rabbitsby Chowdhury et al., Science 254:1802-1805,1991, or in humans by Wilson,Hum. Gene Ther. 3:179-222,1992). Such methods also may be effective fordelivery to various populations of cells in the circulatory or lymphaticsystems, such as erythrocytes, T cells, B cells and haematopoietic stemcells.

[0079] The present invention also provides a composition for deliveringthe nucleic acid construct of the present invention or the TCF responseelement comprising at least 5 TCF binding elements of the presentinvention operably linked to an expressible gene to a cell.

A BRIEF DESCRIPTION OF THE FIGURES

[0080]FIGS. 1a-c show the results of transient transfections of HeLa,HepG2 and SW480 cells with the Tcf responsive luciferase reporterconstruct “5merTcf-SV40-Luc” (CTL501) (FIG. 1a). The numbers indicatethe numbers of base pairs between the Tcf sites. The nucleotide sequenceof 5merTCF-SV40 antisense strand is shown in FIG. 1c. The sequencesunderlined and in italics are active TCF sites. The sequence justunderlined is a mutated TCF site. The sequences in bold are the BglII,NheI and KpnI recognition sites. Cells were transfected with equimolaramounts (about 1 μg each) of the Tcf-responsive and control luciferasereporter constructs as indicated in FIG. 1b. “SV40p” contains the SV40promoter only; “SV40 e/p” contains both the SV40 promoter and enhancer;“CMV” contains the cytomegalovirus enhancer/promoter. Data are expressedas fold activation with respect to the activity of the SV40 promoter setas 1. The mean value and SD from two independent transfections areshown. This result is representative of three independent experiments.

[0081]FIGS. 2a-b show the quantitation of nitroreductase (NTR) expressedby HeLa, HepG2 and SW480 cells infected with CTL102 or with CTL501.Cells were infected at the indicated multiplicities of infection (moi,pfu/cell) with either CTL102, which expresses NTR from the CMVenhancer/promoter, c1 (CTL501), which contains the 5merTcf-SV40-NTRcassette in a left to right orientation or with c13, which contains thesame cassette but in the right to left orientation. Cytoplasmic extractswere prepared two days later and assayed for NTR expression by ELISA(see materials and methods). Infections were done in duplicate. The meanNTR expression level from a representative experiment is shown.

[0082]FIG. 3 shows the antitumour efficacy of CTL501/CB1954 againstHepG2 xenograft tumours in nude mice. Groups of six tumours ranging insize from 20-80 mm² cross sectional area were injected withapproximately 10¹⁰ viral particles as a single injection. Prodrugadministration and tumour measurement are described in materials andmethods.

[0083]FIG. 4 summarises the results of NTR immunostaining ofsubcutaneous SW480 xenografts in nude mice after a single injection ofCTL501 or CTL102 (1.5×10¹⁰ particles in 20 μl of 5% sucrose, 25 mMTris-HCl, pH 7.4). The tumours were excised after 48 hours and monitoredfor NTR expression as described in the materials and methods section.The average percentage NTR positive cells from 3 consecutive sectionsare shown for each dissected tumour.

[0084]FIG. 5 shows a comparison of the in vivo toxicity of CTL501compared to CTL102 following systemic administration and prodrugtreatment. Nude mice were intravenously injected (tail vein) with theindicated number of viral particles. After 48 hours CB1954 was givenintraperitoneally for 5 consecutive days at 20 mg/kg body weight. Thefigure shows the maximum average weight loss (%) during the monitoringperiod (day 1-15) for each animal group (4 animals per group) andpercentage of surviving animals for each treatment group after 15 days.

[0085]FIGS. 6a-c show the results of transient transfections of HeLa andSW480 cells with the Tcf responsive luciferase reporter construct5merTcf-E1BTATA-Luc (CTL502) (FIG. 6a). The nucleotide sequence ofE1BTATA antisense strand is shown in FIG. 6c. The sequence underlinedand in italics is the E1B TATA box. The sequences in bold are theHindIII, BglII, NheI and KpnI recognition sites. Cells were transfectedwith equimolar (about 1 μg each) amounts of several luciferase reporterconstructs as indicated in FIG. 6b. pGL3 basic contains a promoterlessluc cDNA; “E1B” contains the Ad5 E1BTATA box upstream of the luc cDNA;5merTcf-SV40 (CTL501) is described in FIG. 1a. Data are expressed asfold activation compared to the activity of pGL3basic set as 1. The meanvalue and SD of duplicate transfections are shown. The data arerepresentative of three independent experiments.

[0086]FIG. 7 shows, in schematic form, the four different arrangementsof 5 Tcf binding elements that were evaluated in the work described inthis document. For each construct the number of base pairs between thebinding sites is shown, as well as the two dimensional arrangement ofthe sites along the DNA helix based on the assumption that 10.4 bpcorrespond to a complete turn of the helix.

[0087]FIGS. 8a-b show the results of transient transfections of SW480cells using the indicated Tcf responsive luciferase reporter constructs.TcfA, TcfB and TcfC were either combined with the minimal adenoviralE1BTATA box or with the SV40 basal promoter (see FIG. 7 for adescription of TcfA, TcfB and TcfC). The E1B, 5merTcf-SV40 (CTL501),5merTcf-E1BTATA (CTL502) and CMV reporter plasmids are described in thelegend to FIG. 6. Cells were transfected with equimolar amounts (about0.5 μg each) of each luciferase reporter constructs. Data are expressedas percentage activity of 5merTcf-E1BTATA (CTL502) (a) or 5merTcf-SV40(CTL501) (b), respectively. The mean value and SD of triplicatetransfections are shown. These data are representative of threeindependent experiments.

[0088]FIGS. 9a-c shows the results of transient transfections of SW480cells using Tcf-E1BTATA luciferase reporter constructs with differentnumbers and arrangements of Tcf binding sites. FIG. 9a shows the numberof Tcf sites and the spacing between them. FIG. 9c shows the nucleotidesequence of the antisense strands of the Tcf-E1BTATA constructs. Thesequences underlined and in italics are active Tcf sites. The sequencesin bold are the BglII, NheI and KpnI recognition sites. In the4merTcf-E1BTATA construct the BglII site is defective. Cells weretransfected with 0.5 μg of each luciferase reporter construct. Data areexpressed as percentage activity of 5merTcf-E1BTATA (CTL502) (FIG. 9b).The mean value and SD of triplicate transfections are shown. The resultsare representative of three independent experiments.

[0089]FIGS. 10a-c shows the results of transient transfections of SW480cells with TcfC-E1BTATA-luc constructs with variable spacing between theproximal Tcf site and the TATA box (25-499 bp). The structure of theTcfC element is described in FIG. 7. FIG. 10a shows the structure of theTcfC-E1BTATA reporter constructs used for this assay. “d” indicates thenumber of base pairs from the last nucleotide of the proximal Tcfbinding site to the first T of the TATA sequence. The 88 bp and 447 bpspacer fragments were derived by PCR from the human β-globin gene intronII. Cells were transfected with 0.5 μg of each luciferase reporterconstruct. Data are expressed as percentage activity of the TcfC-E1BTATAd=25 construct (b). The mean value and SD of triplicate transfectionsare shown. The results are representative of three independentexperiments. The nucleotide sequence of the antisense strand ofTcfC-E1BTATA when d=25 is shown in FIG. 10c. The sequences underlinedand in italics are active Tcf sites. The sequences in bold are theE1BTATA box, and the SmaI and KpnI recognition sites.

[0090]FIG. 11 shows the quantitation by ELISA of NTR expressed by HeLaand SW480 cells infected with CTL102, CTL501 and CTL502. Cells wereinfected with a range of mois (1, 5, 20,100, 500 and 1500 pfu/cell).Cytoplasmic extracts were prepared two days later and assayed for NTR byELISA (materials and methods). Mean values of duplicate infections areshown.

[0091]FIGS. 12a-b show a comparison of the levels of recombinantadenovirus-directed NTR expression in the livers of normal micefollowing i.v. injection with CTL102 and CTL501. Mice were tail veininjected with the indicated quantities of CTL102 or CTL501, sacrificed48 h later and livers stained for NTR as described in Materials andmethods for tumour sections.

[0092]FIG. 13 shows a comparison of the level of anti-tumour efficacy ofCTL501/CB1954 with CTL102/CB1954 in a xenograft model of colon cancer.Groups of SW480 tumours (n=5) ranging in size from 20-80 mm² crosssectional area were injected with a single dose (10⁹ or 10¹⁰ particles)of either CTL102 or CTL501. Prodrug treatment and tumour measurementwere done as described in Materials and methods.

[0093]FIG. 14 shows a comparison of the level of anti-tumour efficacy ofCTL503/CB1954 with CTL102/CB1954 in a xenograft model of colon cancer.Groups of SW480 tumours (n=5) ranging in size from 20-80 mm² crosssectional area were injected with a single dose (10¹⁰ particles) ofeither CTL102 or CTL503. Prodrug treatment and tumour measurement weredone as described in Materials and methods.

[0094]FIGS. 15a-d show that CTP1 and CTP3 express at very low levels innormal human endothelial cells, dermal fibroblasts and hepatocytes.Cells were infected with Ad.CMV-nLacZ, Ad.CTP1-nLacZ and Ad.CTP3-nLacZas described in Materials and methods and analysed 48 h later. For thedata shown in FIGS. 15a and b, extracts were prepared from cellsinfected with the indicated MOIs (pfu/cell) and beta-galactosidaseassayed using the Galacto-Light assay system as described by thesupplier. Error bars represent standard deviation. A representativeexperiment is shown in each case. In FIG. 15c, cells were infected withan MOI of 1000 pfu/cell and beta-galactosidase-expressing cellsvisualised by X-Gal staining. Only fibroblasts infected withAd.CMV-nLacZ expressed detectable enzyme. FIG. 15d shows that all threeviruses directed high-level beta-galactosidase expression in infectedSW480 colon cancer cells.

[0095]FIGS. 16a-b show that CTP1 and CTP3 express at very low levelsunder replicating conditions in the adenovirus helper cell lines (PerC6and 293). PerC6 and 293 cells were infected at an MOI Of 100 andharvested 30 h later. Cell extracts were then prepared and assayed forβ-galactosidase as described in Materials and methods. Error barsrepresent standard deviation. A representative experiment is shown. FIG.16a has a logarithmic, and 16b a linear, scale.

[0096]FIG. 17 shows that CTP1 and CTP3 express at high levels insecondary colorectal cancer tissue but not in attached liver tissue. 2-3mm³ segments of freshly excised secondary colorectal cancer (liver) withattached liver margin tissue were incubated in the indicated viruses,fixed 48 h later and stained for β-galactosidase expression as describedin Materials and methods. “T” denotes tumour tissue, “L” denotesattached liver tissue.

[0097]FIGS. 18a-b shows that high-level CTP1 and CTP3 expression inprimary colorectal cancer specimens correlates with high-level,non-membranous expression of β-catenin.

[0098] 2-3 mm³ segments of freshly excised primary colorectal cancerwere transduced with the indicated viruses and stained forβ-galactosidase expression as described in Materials and methods.Samples of each tumour were sectioned and stained for β-cateninexpression as described in Materials and Methods.

DETAILED DESCRIPTION OF THE INVENTION

[0099] The invention is described in detail by the use of the followingexamples. These are by way of illustration only and are not to be takenas limiting.

EXAMPLES

[0100] Materials and methods

[0101] Cell Culture

[0102] HepG2 (human liver carcinoma; mutated β-catenin), SW480 (humancolon carcinoma, mutated APC) and HeLa (human cervix carcinoma) celllines were obtained from ATCC and were maintained as recommended by thesupplier. PER.C6 (human embryonic retinoblast cell line) cells wereobtained from IntroGene, (Fallaux et al., Human Gene Ther., 9:1909-1917, 1998) and were cultured in DMEM supplemented with 10% FCS, 2mM MgCl₂ and antibiotics (150 μg/ml penicillin, 250 μg/ml streptomycin).

[0103] Primary human dermal fibroblasts were isolated from punch biopsysamples from healthy volunteers and maintained as above. HUVEC cellswere obtained from Promocell (Heidelberg, Germany) and maintained inEndothelial Growth Medium plus Supplement Mix (Promocell). Primaryhepatocytes were maintained in Williams medium with added antibiotics,glutamine, insulin and hydrocortisone.

[0104] Plasmid Construction

[0105] To clone pGL3pro/5merTcf-SV40 (CTL501) two partiallydouble-stranded fragments were generated by annealing the partlycomplementary pairs of oligonucleotides 1 with 2, and 3 with 4. Oligo 1:PCT AGC AAG CTT ACT AGT CCT TTG ATC AAG AGT CCT ACC (SEQ ID NO:1) TTTGAT CTC TAA ATG CAC CTT TGA TC Oligo 2: PAC TGA ATT CCT TGA TCA AAG GTGCAT TTA GAG ATC AAA (SEQ ID NO:2) GGT AGG ACT CTT GAT CAA AGC ACT AGTAAG CTT G Oligo 3: PAA GGA ATT CAG TCC TTT GAT CAA GAG TCC TAC CTT TGA(SEQ ID NO:3) TCT CTA AAT GCA CCT TTG ATC A Oligo 4: PGA TCT GAT CAA AGGTGC ATT TAG AGA TCA AAG GTA GGA (SEQ ID NO:4) CTC TTG ATC AAA GG (whereP indicates phosphate modification)

[0106] These fragments each contain three TCF binding sites (consensusCCTTTGATC). These two double-stranded fragments were ligated and theresulting fragment (about 130 bp) containing six TCF sites was cloned bytheir NheI and BglII sites into NheI/BglII-digested pGL3 promoterplasmid (pGL3pro; Promega) resulting in pGL3pro/5merTcf-SV40 clone 10.However, sequencing revealed that the most 3′ TCF-binding site containeda deletion of one G nucleotide, producing an inactive site (CCTTTATC)(See FIG. 1c).

[0107] To generate pGL3basic/5merTcf-E1BTATA (CTL502) 84 bp from theClontech plasmid pG5CAT, spanning 21 bp upstream and 55 bp downstream ofthe adenoviral E1BTATA box (TATATAAT), were amplified with PCR usingoligos containing BglII and HindIII overhangs. This fragment, containing16 bp of the Ad5 E1B promoter (GGGTATATAATGCGCC) (SEQ ID NO:11), wasthen cloned into BglII/HindIII-digested pGL3basic resulting inpGL3basic/E1BTATA clone 2 (see FIG. 6c). Then the 5-mer TCF sites frompGL3basic/5merTcf-SV40 (CTL501) clone 10 were cut out by NheI/BglIIdigestion and cloned into NheI/BglII-digested pGL3basic/E1BTATA clone 2,resulting in pGL3basic/5merTcf-E1BTATA clone 1. Sequencing confirmed theexpected construct.

[0108] To generate pGL3pro/TcfA-SV40 clone 4, pGL3pro/TcfB-SV40 clone31, pGL3pro/TcfC-SV40 clone 3, pGL3basic/TcfA-E1BTATA clone 28,pGL3basic/TcfB-E1BTATA clone 10 and pGL3basic/TcfC-E1BTATA clone 1,oligonucleotides 5, 7 and 9, respectively, each containing 5 active TCFbinding sites (CCTTTGATC) and an intentionally mutated site (CCTTTATC,for consistency) were annealed to their respective antisenseoligonucleotides, 6, 8 and 10. Oligo 5: CTA GCA AGC TTA CTA GTC CTT TGATCA AGA GTT TCC TAC (SEQ ID NO:5) CTT TGA TCT CTA AAT TGC ACC TTT GATCAA GGA ATT CAG TCC TTT GAT CAA GAG TAA CCT ACC TTT GAT CTC TAA ATG CACCTT TAT CA Oligo 6: GAT CTG ATA AAG GTG CAT TTA GAG ATC AAA GGT AGG TTA(SEQ ID NO:6) CTC TTG ATC AAA GGA CTG AAT TCC TTG ATC AAA GGT GCA ATTTAG AGA TCA AAG GTA GGA AAC TCT TGA TCA AAG GAC TAG TAA GCT TG Oligo 7:CTA GCA AGC TTA CTA GTC CTT TGA TCA AGC TAO CTT TGA (SEQ ID NO:7) TCTCTA GCA CCT TTG ATC AAG AGT CCT TTG ATC AAG CCT ACC TTT GAT CTC TAA ATGCAC CTT TAT CA Oligo 8: GAT CTG ATA AAG GTG CAT TTA GAG ATC AAA GGT AGGCTT (SEQ ID NO:8) GAT CAA AGG ACT CTT GAT CAA AGG TGC TAG AGA TCA AAGGTA GCT TGA TCA AAG GAC TAG TAA GCT TG Oligo 9: CTA GCA AGC TTA CTA GTCCTT TGA TCA ATA CCT TTG ATC (SEQ ID NO:9) TCA CCT TTG ATC AAG TCC TTTGAT CAT ACC TTT GAT CTC TAA ATG CAC CTT TAT CA Oligo 10: GAT CTG ATA AAGGTG CAT TTA GAG ATC AAA GGT ATG ATC (SEQ ID NO:10) AAA GGA CTT GAT CAAAGG TGA GAT CAA AGG TAT TGA TCA AAG GAC TAG TAA GCT TG (all sixoligonucleotides were modified by 5′ phosphate).

[0109] The resulting fragments containing NheI/BglII overhangs werecloned into NheI/BglII digested pGL3promoter or NheI/BglII digestedpGL3basic/E1BTATA clone 2 to generate the aforementioned constructs. Thesequences were confirmed to be as expected.

[0110] The constructs pGL3basic/2merTcf-E1BTATA clone 9,pGL3basic/3merTcf-E1BTATA clone 2, pGL3basic/3merTcf-E1BTATA clone 13,pGL3basic/4merTcf-E1BTATA clone 15 and pGL3basic/4merTcf-E1BTATA clone34 were cloned by the same way, but, these constructs have one or moreTcf sites deleted most probably due to loop generation during annealingand following excision of loops in E. coli after transformation. Forspacing between the TCF sites and confirmed sequences for theseconstructs see FIGS. 9a and c.

[0111] pGL3basic/88-E1BTATA clone 8 and pGL3basic/TcfC-88-E1BTATA clone2 (distance to TATA box: d=140) were constructed by cloning a PCRamplified 88 bp fragment (5′ oligo: GAAGATCTCCCCTTCTTTTCTATGGTTAAG (SEQID NO:12), 3′ oligo: GAAGATCTGCAATCATTCGTCTGTTTCCC) (SEQ ID NO:13) fromthe human β-globin gene intron 11 using BglII overhangs intoBglII-digested pGL3basic/E1BTATA clone 2 or BglII digestedpGL3basic/TcfC-E1BTATA clone 1, respectively.

[0112] pGL3basic/447-E1BTATA clone 1 and pGL3basic/TcfC-447-E1BTATAclone 6 (distance to TATA box: d=499) were cloned by inserting a PCRamplified 447 bp fragment (5′ oligo: GAAGATCTCCCCTTCTTTTCTATGGTTAAG (SEQID NO:12), 3′ oligo: GAAGATCTGATTTGG TCAATATGTGTACAC) (SEQ ID NO:14)from the human β-globin gene intron II using BglII overhangs intoBglII-digested pGL3basic/E1BTATA clone 2 or BglII-digestedpGL3basic/TcfC-E1BTATA clone 1. The sequences were confirmed.

[0113] To create pGL3basic/TcfC-25-E1BTATA clone 6 (distance to TATAbox: d=25), the TcfC fragment was cut out by HindIII/BglII digest frompGL3basic/TcfC-E1BTATA clone 1 and then blunted with mung bean nuclease(NEB). The blunted fragment was cloned into a partially XbaI-digested,mung bean nuclease-blunted pGL3basic/E1BTATA clone 2, to create theintermediate construct pGL3basic/TcfC-25-E1BTATA clone 19. From here, wehad to reclone the TcfC-25-E1BTATA promoter fragment into pGL3basic asthe plasmid backbone immediately upstream of the promoter was changedfor unknown reasons in clone 19. Therefore the TcfC-25-E1BTATA promoterwas cut out by SpeI/HindIII digest from pGL3basic/TcfC-25-E1BTATA clone19 and then blunted with Klenow enzyme. Finally, the bluntedTcfC-25-E1BTATA fragment was cloned into HindIII digested andKlenow-blunted pGL3basic to generate pGL3basic/TcfC-25-E1BTATA clone 6(d=25) with the correct plasmid backbone. The sequence was determinedsince the blunting process is error-prone (see FIG. 10c)

[0114] Transient Transfections and Luciferase Assay

[0115] HepG2, SW480 and HeLa were seeded the day before transfection atdensities of 2.5×10⁵, 1.5×10⁵ and 6.0×10⁴ cells per 6-well respectively.The next day, a mixture of CL22 peptide(KKKKKKGGFLGFWRGENGRKTRSAYERMCNILKGK (SEQ ID NO:15) (described inInternational Patent Application WO 98/35984)) and plasmid DNA (2:1ratio, pg:pg) was prepared in a final volume of 100 μl in HBS (10 mMHepes pH 7.4, 150 mM NaCl; Sigma) and incubated at RT for 30-45 minbefore addition of 0.9 ml of “RAC” solution (0.1% human albumin (BPL,UK), 120 μM chloroquine (Sigma) in RPMI medium (Sigma). The transfectionsolution was then added to cells after washing them once with PBS andincubated with the cells for 4-5 hours before replacement with 2 ml offresh complete medium. After two days, cells were washed once with PBSand then incubated for 10 min at RT in 200 μl lysis buffer (10 mM sodiumphosphate pH 7.8, 8 mM MgCl₂, 1 mM EDTA pH 8.0,1% Triton X-100 and 15%glycerol). After centrifugation, an aliquot of the supernatant wasassayed for luciferase activity with luciferase assay buffer (0.1 mMluciferin, 0.44 mM ATP in lysis buffer) using a luminometer (Lumat LB9501, Berthold, Germany). Activity was normalised using the proteincontent of each sample (BCA, Pierce).

[0116] Construction of Replication Defective Adenoviruses Expressing NTR

[0117] The transfer vectors used to construct the recombinantadenoviruses c1/CTL501, c13 and CTL502 i.e. pPS1128/5merTcf-SV40 (clones1 and 13) and pPS1128/5merTcf-E1BTATA (clone 10) respectively wereconstructed in two stages. In the first, the 5merTcf-SV40 promoter frompGL3pro/5merTcf-SV40 clone 10 and the 5merTcf-E1BTATA promoter frompGL3basic/5merTcf-E1BTATA clone 1 were cloned as Hind III fragments intoHindIII digested pTX0374 resulting in replacement of the CMVenhancer/promoter with the respective Tcf promoters to create“pTX0374/5merTcf-SV40 clone 8” and “pTX0374/5merTcf-E1BTATA clone 1”.pTX0374 contains a CMV-NTR-IVSII-p(A) (NTR: E. coli B/r nitroreductasegene amplified from genomic DNA) expression cassette with the humanβ-globin intron 11 for transcriptional stabilisation and the Complement2 gene poly(A) signal for termination and the plasmid pBluescript KS+ asbackbone. In the second stage, the complete expression cassettes frompTX0374/5merTcf-SV40 clone 8 and pTX0374/5merTcf-E1BTATA clone 1 wererecloned into SpeI digested pPS1128 to create “pPS1128/5merTcf-SV40clones 1 and 13” (clone 1: left to right orientation in E1; clone 13:right to left orientation in E1) and “pPS1128/5merTcf-E1BTATA clone 10(left to right orientation in E1). pPS1128 was kindly provided by Dr. P.Searle, CRC Institute of Cancer Studies, University of Birmingham.pPS1128 contains adenoviral sequences from the left hand ITR to nt 359and from nt 3525 to 10,589 and is therefore an E1-deleted vector.

[0118] pTX0375, the transfer vector used to generate CTL102, wasconstructed by cloning a SpeI fragment spanning the whole expressioncassette (CMT-NTR-IVSII-p(A)) from pTX0374 into SpeI digested pPS1128and identification of a clone containing the cassette in the left toright orientation.

[0119] The adenoviral “backbone” vector pPS1160 was constructed by PacIlinearisation of pPS1128, ligation with a PacI-compatible adaptor(oligo1: 5′-TACATCTAGATAAT (SEQ ID NO:16)-3′, oligo2: 5′-TTATCTAGATGTA(SEQ ID NO:17)-3′) containing an XbaI site followed by XbaI digestion torelease a ca. 7 kb XbaI fragment containing Ad5 sequences 3524-10589.This was then cloned into XbaI linearised pPS1022 (Dr. Peter Searle) apUC18-based plasmid containing Ad5 sequences from nt 10,589 to the righthand ITR but lacking nt 28,592 to 30,470 (E3 region).

[0120] The recombinant viruses CTL501, CTL502 and CTL102 wereconstructed by homologous recombination in Per.C6 cells. These werecotransfected with an equimolar mixture of pPS1128/5merTcf-SV40 (clone 1or clone 13), pPS1128/5merTcf-E1BTATA (clone 10) or pTX0375,respectively, and pPS1160 into 90% confluent PER.C6 cells. Therecombinant viruses were harvested about 7 days later by 3 freeze-thawcycles in infection medium (DMEM, 1% FCS, 2 mM MgCl₂). By repeatedinfection/harvesting cycles the viruses were grown to large scale andthen purified by standard CsCl density centrifugation, dialysed againstexcess of storage buffer (10 mM Tris pH 7.4,140 mM NaCl, 5 mM KCl, 0.6mM Na₂HPO₄, 0.9 mM CaCl₂, 0.5 mM MgCl₂ and 5% sucrose) and finallysnap-frozen in liquid nitrogen and stored at −80° C. Particleconcentrations were determined using the BCA Protein Assay Reagent(Pierce). Infectious titres were either estimated on the assumption that1 in 100 particles are infectious or were determined in a limitingdilution standard method against a defined internal virus standard.Adenoviral DNA was characterised by restriction digestion and directsequencing using viral DNA as a template (the promoter region up to thestarting NTR reading frame was sequenced).

[0121] pTX0374 was constructed by cloning a 1.6 kb BglII-BamHI fragmentcontaining the CMV promoter fused to the E. coli ntr gene into pSW107.This plasmid was constructed by cloning a 917 bp fragment of the humanbeta-globin gene (BamHI site in exon2 to the EcoRI site in exon3)coupled to a 240 bp HincII-BamHI fragment containing the polyA additionand transcriptional termination signals of the human complement C2 geneinto pBluescript (Stratagene). pTX0375 was constructed by cloning a 2.5kb SpeI fragment from pTX0374 into SpeI-digested pPS1128. This plasmidwas constructed in two stages. In the first, the left hand EcoRI site ofpPS971 (Weedon et al, Int. J. Cancer, in press) was converted to a SwaIsite to create pPS115. In the second, the 350 bp Spe1-AflII fragment ofpPS115 was replaced with a linker prepared by annealing the twooligonucleotides: (SEQ ID NO:19)-3′ 5′-CTAGTATCGATTGTTAATTAAGGGCGTGGCCand (SEQ ID NO:20)-3′ 5′-TTAAGGCCACGCCCTTAATTAACAATCGATA.

[0122] pPS1022 was constructed from pPS972 by conversion of the righthand EcoRI site to a SwaI site.

[0123] It will be appreciated by those skilled in the art that anysuitable vector may be used in the construction of the Tcf responsiveelement of the present invention. In particular, it will be appreciatedthat any suitable adenovirus based vector can be used in theconstruction of the Tcf responsive element of the present invention.

[0124] Preparation of Adenoviral DNA

[0125] Viral DNA was prepared from about 10¹¹ CsCl-banded virusparticles by incubation with 100 μg/ml proteinase K in 20 mM Tris/HCl pH7.5, 5 mM EDTA pH 8.0, 0.1% SDS for 3-4 hours at 37° C. The crude DNApreparation was then extracted two times with an equal volume ofPhenol:Chloroform:Isoamylalcohol (25:24:1), once with chloroform onlyand then precipitated with {fraction (1/10)} Vol. 3.0 M Na Acetate and 2Vol. 100% Ethanol in a dry ice/ethanol bath for 10 min. Aftercentrifugation, (10 min at 13K at RT) the resultant DNA pellet waswashed with 70% Ethanol, air-dried and then resuspended in water. ViralDNA was analysed by restriction digestion. The promoter regions ofCTL501 and CTL502 were analysed by DNA sequencing (Seqlab GmbH,Germany).

[0126] Assay of NTR Expressed by Virus Transduced Cells (ELISA)

[0127] 1-1.5×10⁴ cells per 6-well cell culture plate were transduced byincubation with virus in infection medium (DMEM/1% FCS) for 90 min at37° C. in a 5% C02 atmosphere followed by incubation in complete mediumfor 2 days.

[0128] Cytoplasmic extracts were then prepared by hypotonic lysis. Cellswere washed with PBS before lysis with ice-cold hypotonic lysis buffer(10 mM Tris pH 7.5) for 45 min on ice. Extracts were cleared bycentrifugation at 13K for 2 min. 96-wells (Nunc-Immuno Plate MaxisorpAssay Plates) were coated overnight in triplicate with 50 μl extract.Wells were washed three times with PBS/0.5% Tween20 and then incubatedwith sheep anti-NTR polyclonal antibody (100 μl of 1:2,000 dilution inPBS/0.5% Tween20) for 30 min at RT. After removal of excess primaryantiserum by washing 3× with PBS/0.5% Tween20 the extracts wereincubated with donkey anti-sheep HRP conjugate (100 μl of 1:5,000dilution in PBS/0.5% Tween20) for 30 min at RT. After three washes withPBS wells were incubated with 100 μl of a solution prepared by mixing 1ml of TMB (1 mg/ml in DMSO; Sigma) with 9 ml 0.05 M phosphate-citratebuffer (Sigma) and 2 μl of 30% (v/v) H₂O₂. Wells were incubated for 15min at RT and the reaction stopped with 25 μl of 2M H₂SO₄. OD 450 nm wasread using a 96-well plate reader (Labsystems Multiscan MS).

[0129] NTR Immunostaining of Virus-Injected Xenografts

[0130] SW480 tumours were injected with 20 μl of either CTL102 or CTL501and excised 48 hours later following humane sacrifice. The tumours werethen fixed in 4% formalin/PBS for 20-24 hours at 4° C. before embeddingin paraffin wax (Citadel 2000). 3-4 μm wax sections were cut andcollected on APES-treated glass slides. Sections were dewaxed,rehydrated and washed 2×in PBS/0.01% Tween20 (5 min) and then immersedin 0.25% H₂O₂/PBS for 30 min at RT. Sections were then washed 3×inPBS/0.01% Tween20 (5 min) and then permeabilised with ice cold 0.1%Triton-X100 for 5 min at RT followed by 2×washing for 5 min in PBS/0.01%Tween200. Sections were then blocked with 5% normal rabbit serum in PBSfor 60 min at RT before incubation with a polyclonal sheep anti-NTR(1:2,000 diluted in PBS) for 60 min at RT. Excess primary antibody wasremoved by washing 3× with PBS/0.01% Tween20 (5 min) before incubationwith a biotinylated anti-sheep IgG/Streptavidin-HRP solution (VectastainABC kit, Vector Laboratories, PK-6106, 1:200 diluted in PBS) for 30 minat RT.

[0131] Sections were washed 3× for 5 min with PBS/0.01% Tween20 and thenincubated with freshly prepared AEC reagent (Vectastain, VectorLaboratories). Reactions were stopped after 10 min by washing in waterand finally sections were mounted in an aqueous mountant. Staining wasanalysed by counting the number of positively stained cells within astandardised area.

[0132] Generation of Subcutaneous Tumour Xenografts in Nude Mice

[0133] Tumour xenografts were generated by subcutaneous injection of oneflank of male Balb/c nu/nu mice (6-8 weeks old, Harlan UK) with 100 μlof a suspension of exponentially growing cultured tumour cells, washedand resuspended in sterile saline solution. Cell viability was at least90%. For HepG2 an innoculum of 5×10⁶ cells was used. For SW480 this wasslightly lower at 2×10⁶ cells. Following injection, mice were kept in asterile environment and examined regularly for the appearance of tumourxenografts.

[0134] Intratumoural Injection of CTL102 and CTL501

[0135] A U-100 insulin syringe (TERUMO, Leuven, Belgium) fitted with afixed 27-gauge needle was used to inject 20 μl of virus suspension orvehicle alone (5% sucrose in 25 mM Tris-HCl, pH 7.4) directly intotumours through the skin. To avoid virus leakage, injections wereperformed in a continuous slow movement and the needle was held in placefor about 15 seconds after injection was completed.

[0136] CB1954 Treatment Schedule and Tumour Size Measurements

[0137] CB1954, freshly dissolved in DMSO and diluted 1 in 5 with NSS,was administered by intraperitoneal injection. Mice received 5consecutive daily doses of 20 mg/kg body weight. Mice treated with thevehicle alone were injected with 20% DMSO/saline (5.0 μl/kg bodyweight). Tumour growth was monitored by measuring the tumour diameterthrough the skin in two perpendicular dimensions (length and width)using callipers and expressed as surface area (length×width=mm²). Toprevent undue suffering compulsory sacrifice was carried out when thetumour reached 140 mm². Weight loss and changes in animal behaviour(signs of distress) were also recorded.

[0138] Intravenous Injection of CTL501/CTL102 and CB1954 Administrationin Nude Mice

[0139] A syringe fitted with a fixed 27-gauge needle was used toadminister 100 μl of virus suspension into the tail vein of nude mice.After 48 hours, CB1954 was administered as described above. Mice weremonitored and weighed daily. To prevent undue suffering to animalshumane sacrifice was carried out if mouse body weight was reduced bymore than 20% or at the onset of any sign of severe distress.

[0140] Ex Vivo Transduction of Freshly Excised Colorectal Tumour Samples

[0141] Freshly excised tumour tissue was extensively washed (at least 10minutes duration) under aseptic conditions with 20 ml of Earl's MEMcontaining 10% FCS, 150 μg/ml penicillin, 250 μg/ml streptomycin, 10μg/ml tetracycline, 100 μg/ml amikacin, 150 g/ml chloramphenicol and 100μg/ml gentamycin and stored at 4° C. overnight in medium containing 10%FCS. After removal of fat and grossly and suspected necrotic tissue, 2-3mm³ samples were prepared and placed individually into wells of a96-well plate. Samples (in quadruplicate) were incubated in 150 μl ofserum-free medium containing, 1.0×10¹⁰ virus particles or in mediumalone for 4 h in a CO₂ incubator. The medium was then replaced with EMEMcontaining 10% FCS and the samples incubated for a further 44 h to allowgene expression to proceed. β-galactosidase expression was visualisedafter fixation of the tumour samples in 2% paraformaldehyde/PBS for 2 hat 4° C. and washing with PBS by overnight incubation in X-gal stainingsolution at 37° C.

[0142] β-Catenin Immunohistochemical Staining

[0143] Paraffin-embedded sections were stained for β-catenin usingrabbit polyclonal antiserum (Santa Cruz Biotechnology Inc) and vectorAEC kit.

[0144] Construction of CTL503, Ad.CTP1-nLacZ and Ad.CTP3.nLacZ

[0145] The transfer vector for CTL503 was constructed by cloning theTCFC_(d=25)-E1BTATA promoter as a HindIII fragment upstream of the NTRgene in HindIII-digested pTX0374, so removing the CMV promoter from thelatter. The new expression cassette was cloned into pPS1128 as a SpeIfragment to create pPS1128/TCFC_(d=25)-E1BTATA.

[0146] The transfer vector for Ad.CTP1-nLacZ was constructed by cloningthe an expression cassette comprising of a nuclear-targeted LacZ genefused to the mouse protamine polyadenylation signal as an XbaI fragmentinto XmaI/SpeI-digested and blunted pTX0374. The CTP1 promoter wascloned upstream of the nLacZ gene as a HindIII fragment The CTP1-nLacZexpression cassette was then cloned in a left-to-right orientation intopPS1128 as a blunted, SpeI/NotI fragment into SpeI, blunted pPS1128 tocreate pPS1128/CTP1-nLacZ.

[0147] To construct the transfer vector for Ad.CTP3-nLacZ, in a firststep the CTP3 promoter was cloned as a blunted, SpeI/HindIII fragmentinto HindIII-digested and blunted pTX0374, replacing the CMV promoterwith CTP3. This cloning regenerated the HindIII site. In a second step,the resulting plasmid was digested with HindIII/PacI and then blunted torelease the NTR gene and the IVSII intron/polyadenylation signal. ThenLacZ-poly(A) cassette (see details described for the construction ofCTP1-nLacZ) was cloned downstream of CTP3 as a blunted XbaI fragment.Finally the complete expression cassette was cloned into pPS1128 in aleft-to-right orientation as a blunted, SpeI/NotI fragment into the PmeIsite of a pPS1128 derivative (pTX0398) in which the unique SpeI site isreplaced by a PmeI site to create pTX0398/CTP3-nLacZ.

[0148] Viruses were rescued by homologous recombination of the abovetransfer vectors with pPS1160 in PerC6 cells as described above(Construction of replication defective adenoviruses expressing NTR)

[0149] X-Gal Staining of Cells for Histology

[0150] After washing cells with PBS, cells were fixed in 0.05%glutaraldehyde in PBS for 10 min at RT. Following further washing inPBS, cells were incubated cells the following solution and incubated at37° C. X-gal solution: 400 μl 500 mM K₄Fe(CN)₆, 400 μl 500 mM K₃Fe(CN)₆,100 μl 2 mM MgCl₂, 250 μl 40 mg/ml X-Gal in DMF and 8.85 ml PBS.

[0151] Quantitation of β-Galactosidase Expression

[0152] Expression of β-galactosidase was measured for quantitativegraphical presentation by means of the Galacto-Light system (Tropix Inc,Applied Biosystems, Foster City, Calif., U.S.A.) EXAMPLES

Example 1

[0153] A 5merTcf-SV40-Luciferase Construct is Specifically Activated inTumour Cell Lines with Deregulated β-Catenin Activity.

[0154] To evaluate the ability of β-catenin/Tcf binding elements todirect high-level gene expression specifically in cells with deregulatedβ-catenin activity we constructed a luciferase reporter plasmidcontaining an artificial promoter comprising of 5 Tcf sites upstream ofthe basal SV40 promoter. In HeLa cells (β-catenin not deregulated) the5merTcf-SV40 was not more active than the SV40 promoter alone. Incontrast, in cell lines with deregulated β-catenin the 5merTcf-SV40promoter expressed at about 80% activity of the CMV enhancer/promoter(SW480 cells) and was even more active than CMV (HepG2). The inductionratios for the 5merTcf-SV40 were 18 (HepG2) and 44.2 (SW480). These dataindicate that the Tcf sites are active only in the presence of nuclearβ-catenin. In some experiments the activity of the 5merTcf-SV40 promoterin Hela cells was even lower than the activity of SV40 alone (data notshown). This is consistent with the fact that Tcf factors normallyrepress transcription e.g. by interacting with the transcriptionalcofactor CBP (cAMP binding protein) if β-catenin is not present tocreate β-catenin/Tcf heterodimers.

Example 2

[0155] The activity and Specificity of the 5merTcf-SV40 ArtificialPromoter is Retained in a Replication Defective Adenovirus Vector.

[0156] To evaluate the utility of the 5merTcf-SV40 promoter to drive thehigh-level expression of a therapeutic gene selectively in tumourscomprising of cells with deregulated β-catenin, for instance a tumour ofcolorectal origin, we constructed a replication defective Adenovirusvectors expressing the E. coli B nitroreductase gene (NTR) under thecontrol of the 5merTcf-SV40 promoter. The NTR gene encodes an enzymethat can convert the prodrug CB1954 into a potent DNA cross-linkingagent that can kill both dividing and non-dividing cells. Clones 1(CTL501) and 13 contain the cassette in the indicated orientation (FIG.2a). In CTL102, the CMV enhancer/promoter regulates NTR expression.CsCl-banded viruses were prepared and HeLa, HepG2 and SW480 cells wereinfected with the indicated mois (FIG. 2b). In this case theleft-to-right orientation of clone 1 was found to offer slightly greaterspecificity of expression, as shown, and this became the standardorientation adopted (CTL501). As expected CTL102 showed NTR expressionin all three cell lines independent of their β-catenin status. Incontrast, CTL501 was highly active only in HepG2 and SW480 cells. Evenat a 5× higher moi NTR expression was barely detectable in HeLa cells.Clone 13 whilst as active as CTL501 in HepG2 and SW480 cells alsoexpressed in Hela although still at a relatively low level compared tothe former.

Example 3

[0157] The Tcf-Responsive Adenovirus CTL501 Shows Anti-Tumour ActivityIn Vivo.

[0158] Having established that CTL501 can express high levels of NTR inpermissive cells in vitro we tested whether intratumoral injection ofHepG2 xenografts with the virus resulted in the expression of sufficientenzyme to sensitise the tumours to CB1954 and cause measurableanti-tumour effects including tumour regression. In the experiment 4 outof 5 tumours underwent clear regression (FIG. 3). After 56 days two ofthe four responders could be categorised as complete regressions and twoanimals harboured a quiescent, very small tumour. Tumours injected withvehicle only and treated with CB1954 grew out as expected. Interestinglyvirus injection alone resulted in an apparent slowing of tumour growth.We attribute the variable response to the treatment to the inherentvariability of the intratumoral injection technique.

Example 4

[0159] High Level Expression of NTR in CTL501-Injected SW480 Xenografts.

[0160] Having demonstrated that CTL501 is highly active in SW480colorectal cancer cells in vitro we determined whether high level NTRexpression could be obtained by intratumoral injection of subcutaneousSW480 xenografts in nude mice. Four tumours were injected with eitherCTL102 or with CTL501 and 48 hours later, following humane sacrifice,were excised, fixed, sectioned and immunostained for NTR expression. Theresults are summarised in FIG. 4. These provide further evidence thatCTL501 expresses NTR at a level at least comparable to CTL102. Weattribute the significant variation in the percentage of cells that areNTR positive to the inherent variability of the intratumoral injectiontechnique.

Example 5

[0161] Systemic Administration of Adenovirus Followed by CB1954Treatment: CTL501 is Much Less Toxic than CTL102 (CMV-NTR).

[0162] We show above that intratumoral injection of CTL501 results inhigh level NTR expression. Associated with this method of obtainingspecific delivery of a therapeutic gene to tumour cells however is thedanger of virus dissemination, in particular via the bloodstream to theliver. Based on the in vitro specificity data presented above wepredicted that whereas intravenous injection of nude mice with CTL102would result in high level expression of NTR in the liver andconsequently significant toxicity of CB1954, injection of CTL501 wouldbe relatively very well tolerated as this should result in very littleor no liver expression of NTR. The results shown in FIG. 5 support this.Whereas a dose of 10E9 particles of CTL501+CB1954 treatment resulted invirtually no toxicity, a tenfold lower dose of CTL102 resulted in 100%mortality. We conclude that CTL501 expresses no, or an insignificantamount of, NTR in normal cells, in particular the liver.

Example 6

[0163] Construction of an Improved Tcf-Based Promoter that is FullyInactive in Cells Lacking β-Catenin Activity.

[0164] For some applications it would be desirable to increase thespecificity of the 5merTcf-based artificial promoter further, forinstance to control the replication of a therapeutic adenovirus. Wetherefore evaluated the combination of the 5merTcf element describedabove with the adenoviral (Ad5) E1B gene TATA box (5merTcf-E1BTATA-Luc(CTL502), FIG. 6a). As shown above, in transient transfections in HeLaand SW480 the 5merTcf-SV40 promoter is highly active in SW480, herecomparable to CMV, but shows a background activity in HeLa cells due tothe basal activity of the SV40 promoter. In contrast, the5merTcf-E1BTATA-luc construct although less active in SW480 (60%activity of 5merTcf-SV40) was completely inactive in HeLa (notderegulated β-catenin). Expressed another way, replacement of the SV40minimal promoter with the E1BTATA element resulted in an increase ininducibility in SW480 compared to HeLa from about 30-60fold for5merTcf-SV40 to about 600-2,000fold for 5merTcf-E1BTATA i.e. a 20foldimprovement.

Example 7

[0165] The Activity of the 5merTcf-S V40 and 5merTcf-E1BTATA PromoterConstructs is Dependent on the Relative Positioning of the Tcf Sites.

[0166] To determine whether changing the arrangement of the sites alongthe DNA helix influences the activity of 5merTcf-based promoterconstructs we constructed “TcfA”, “TcfB” and “TcfC” (FIG. 7) incombination with either the SV40 minimal promoter or the E1BTATA andcompared their activity with the respective original 5mer Tcf-basedpromoters i.e. +SV40 or +E1BTATA. In transiently transfected SW480cells, the highest level of reporter gene expression was obtained withthe TcfC-E1BTATA promoter element (FIG. 8a). The lowest level ofexpression was obtained with the E1BTATA-TcfB combination which wasabout half as active as TcfC-E1BTATA. For the SV40 combinations lessvariation in expression was observed (FIG. 8b).

[0167] Furthermore in marked contrast to the result with E1BTATA, theTcfC-SV40 promoter was the least active and the TcfB-SV40 the mostactive. These results suggest that the optimum spacing of the Tcf sitesis dependent on the basal promoter that is combined with the Tcfelements. Further evidence for a critical role in the spacing of Tcfsites in determining the level of expression in β-catenin deregulatedcells is provided in FIG. 9. This shows the results of transienttransfections of SW480 with Tcf-E1BTATA-luc constructs containing fewerthan 5 Tcf sites (2, 3 and 4). Whilst there is a general increase inexpression with an increasing number of sites, the results also showthat by appropriate spacing of 3 sites a higher level of expression wasobtained compared to an alternative arrangement of 4 sites.

[0168] None of the constructs described above exhibited an increasedbackground expression level in Hela compared to the original5merTcf-based promoters i.e. alteration of the spacing of the Tcf sitesdoes not result in a loss of the exquisite specificity we have describedabove.

Example 8

[0169] The Activity of the TcfC-E1BTATA Promoter Element is Determinedby the Distance Between the Tcf Binding Sites and the E1BTATA Box.

[0170] We also determined the effect on expression of changing thedistance between the Tcf binding sites and the TATA box (FIG. 10). Fourconstructs based on the TcfC-E1BTATA were constructed and compared (FIG.10a—and see the materials and methods section). As shown in FIG. 10b aninverse relationship was discovered between the Tcf-to-TATA separationand the level of luciferase expression in transfected SW480 cells.Although expression may be further improved by reducing the separationfurther it is likely that 25 bp is approaching the minimum separation.As below this the initiation complexes and Tcf/β-catenin would beexpected to sterically hinder access to their respective binding sites.The new constructs tested here retained the specificity of the original5merTcf-E1BTATA artificial promoter (data not shown).

Example 9

[0171] The Activity and Specificity of the 5merTcf-E1BTATA Promoter isalso Retained in a Replication Defective Adenovirus.

[0172] To confirm that the 5merTcf-E1BTATA (CTL502) promoter alsoretains its activity and specificity in the context of adenovirus weinfected HeLa and SW480 cells with CTL102, CTL501 and CTL502 and assayedNTR expression by ELISA (FIG. 11). As expected, CTL102 was highly activein both HeLa and SW480. Consistent with transient transfection studiesshown above, both CTL501 and CTL502 were highly active in SW480 but onlyvery weakly active in HeLa. Furthermore, CTL502 was apparently lessactive than CTL501 in HeLa. Whereas CTL501 expressed a clearlydetectable level of NTR at high moi (1500 pfu/cell) this was notdetected with CTL502. The combination of β-catenin/Tcf responsiveelements with the adenoviral (Ad5) E1BTATA box thus provides anextremely high level of tumour selectivity likely to be suitable for theexpression of genes encoding highly potent therapeutic agents whichcould significantly damage non-cancerous cells even at low levels.

Example 10

[0173] Intravenous Injection of CTL501 and CTL 102 into Normal Mice:Lack of Liver Expression with CTL501

[0174] We show above (Example 5) that systemic administration of CTL501to normal mice (tail vein injection) followed by CB1954 treatment isvery well tolerated. In contrast, injection of a tenfold lower dose ofCTL102 (CMV.NTR)/CB1954 combined with CB1954 was lethal in all cases. Weconcluded from this result that the β-catenin/Tcf-responsive promoterdriving the NTR gene in CTL501 is inactive or at most weakly active innormal mouse tissues infected by the virus, principally the liver. Toprovide direct evidence for this, mice were injected with CTL501 orCTL102 and liver expression determined by immunostaining 48 hpost-injection. In FIG. 12 we show representative liver sections stainedfor NTR expression. Injection of CTL501 resulted in sporadic, low-levelNTR expression whereas a 10 fold lower dose of CTL102 generated highlevel expression in a majority of cells. We interpret this result andthat described in Example 5 to indicate that the β-catenin/Tcf-4 complexis either absent from or present at too low a level in the nuclei ofnormal mouse hepatocytes to activate the promoter. It is highly unlikelythat these data reflect an inability of murine beta-catenin/Tcf-4 toactivate the promoter as (i) the Wnt pathway is evolutionarily highlyconserved from flies upwards and (ii) the promoter of at least onemurine gene that is activated by Wnt signalling (cdx) contain Tcfbinding sites that fit the consensus human Tcf-4 binding site that wehave used to build the promoter (Lickert et al (2000) Development 127:3805-3813).

Example 11

[0175] CTL501I/CB1954 Anti-Tumour Efficacy in a Xenograft Model ofColorectal Cancer

[0176] We show above in Example 3 that i.t. injection of HepG2 (livercancer) xenografts with a single dose of CTL501 strongly sensitised thisβ-catenin—deregulated tumour model to the prodrug CB1954, resulting intumour regression in the majority of cases. We show here thatCTL501/CB1954 therapy is highly effective in a xenograft model ofβ-catenin-deregulated colorectal cancer (SW480). Two size randomisedgroups of tumours were injected with 10⁹ and 10¹⁰ particles of CTL501respectively and CB1954 administered to the host mice beginning 48 hlater. So as to be able to compare the efficacy observed to thatachievable when NTR was expressed from the CMV promoter, two additionaltumour groups were injected with equivalent doses of CTL102. As shown inFIG. 13, CTL501 and CTL102 injection resulted in a similar level ofanti-tumour efficacy. Non-virus injected control tumours (vehicleinjected+systemic CB1954 treatment) grew strongly.

Example 12

[0177] CTL503/CB1954 Anti-Tumour Efficacy in a Xenograft Model ofColorectal Cancer

[0178] We show above (Examples 6-8) that it is possible to build aβ-catenin/Tcf-4 responsive promoter with an increased dependence onβ-catenin/Tcf-4 (and therefore improved specificity for tumours withderegulated P-catenin) by substitution of the SV40 minimal promoterfragment with the Ad5 E1B TATA and by altering the spacing between theTcf binding sites and between the E1B TATA and promoter-proximal Tcf-4binding site. A recombinant virus (“CTL503”) was constructed containingthis modified promoter driving NTR to determine whether the high levelexpression in permissive cells observed using transient transfectionexperiments would be retained in the context of an adenovirus backbone.In FIG. 14 we show that i.t. injection of SW480 xenografts with CTL503and subsequent CB1954 administration resulted in an anti-tumour responseof a similar magnitude to that resulting from CTL102+CB1954 treatment.These in vivo efficacy data thus provide strong evidence that theimproved tumour specificity of CTP3, detected by transient transfectionexperiments was gained with retention of a high level of activity intumour cells with deregulated β-catenin/Tcf-4.

Example 13

[0179] The β-Catenin/Tcf-Dependent Promoters Express at Very Low Levelsin Cultured Primary Human Hepatocytes, Dermal Fibroblasts andEndothelial Cells

[0180] We show above that the claimed promoters express at very lowlevels in tumour cell lines that retain normal p-catenin regulation.From this we infer that these promoters will be inactive/weaklyexpressed in normal human cells, i.e. we used the tumour cells to modelnormal cells. To provide direct evidence for this we determined theactivity of two of the promoters in a panel of cultured primary humancells (hepatocytes, endothelial cells and dermal fibroblasts). Tofacilitate these and subsequent studies involving cultured primary humantissue (see Examples 15 and 16 below) we constructed recombinantadenoviruses expressing beta-galactosidase under the control of two ofthe claimed promoters, “Ad.CTP1-nLacZ” and “Ad.CTP3-nLacZ” (in theinterest of clarity we now use a systematic nomenclature for thepromoters: the original promoter present in CTL501 is renamed as “CTP1”;the optimised promoter present in CTL503 is renamed as “CTP3”).

[0181]FIG. 15 shows that the CMV promoter expressed strongly in allthree cell types tested whereas CTP1 and CTP3 directed very low levelsof beta-galactosidase expression in these cells. All three promoterswere however strongly active in SW480 colon cancer cells (FIG. 15d)

Example 14

[0182] CTP1 and CTP3 Direct Very Low Levels of Transgene ExpressionDuring Growth of an E1-Deleted Adenovirus in the 293 and PerC6 HelperCell Lines

[0183] Attempts to grow E1-deleted viruses encoding cytotoxic genesdriven by promoters that are active in E1-expressing Ad helper cells aregenerally unsuccessful as expression of the toxic gene product preventsthe cells from supporting efficient virus growth.

[0184] To determine the level of expression of the CTP1 and CTP3promoters during virus production we infected 293 and PerC6 cells withAd.CMV-nLacZ, Ad.CTP1-nLacZ and Ad.CTP3-nLacZ viruses and determined thelevel of LacZ expression 30 h post-infection.

[0185] In FIG. 16 we show that LacZ expression driven by CTP1 and CTP3was significantly lower than that driven by the CMV promoter in both Adhelper lines. However, as observed in all other non-permissive celllines, CTP3 was clearly less active than CTP1 (approximately 10 fold).Both promoters were approximately 3 fold more active in PerC6 than in293 cells. These data suggest that both cell lines but in particular 293cells would support the efficient replication of first generationAdenovirus vectors encoding a toxic transgene under the control of theCTP3 promoter.

Example 15

[0186] CTP1 and CTP3 Promoters are Highly Active in Freshly ExcisedMetastatic Colorectal Cancer Tissue but Inactive in Associated LiverTissue

[0187] Established tumour cell lines are useful model systems to studygene expression patterns in cancer. These lines are however cloned fromprimary cancers, which are polyclonal populations of genetically diverseand genetically unstable cells. They have also generally beencontinuously cultured for long periods of time, providing further scopefor the selection of cells that are well adapted to ex vivo culture butnot very representative of the cancer from which they were derived. Forthese reasons we determined the activity of CTP1 and CTP3 in freshlyexplanted samples of primary and secondary colorectal cancer. Sampleswere prepared and infected with Ad.CMV-nLacZ, Ad.CTP1-nLacZ andAd.CTP3-nLacZ viruses and analysed for nuclear beta-galactosidaseexpression as described in the Materials and methods section.Ad.CMV-nLacZ was used to determine the viability of each sample andsusceptibility to adenovirus infection and to allow a comparison of therelative activities of CMV and CTP1/3 promoters. FIG. 17 shows theresults obtained with a secondary cancer isolated from the liver. Foreach virus treatment, tumour with attached liver margin was incubated inthe virus suspension. As observed with all other tumour specimens, thetissue was free of endogenous beta-galactosidase activity (mock-infectedsamples not stained by X-Gal). Incubation with Ad.CMV-nLacZ resulted instrong staining of both tumour and attached liver. A strikingdemonstration of tumour specificity was provided by infection ofequivalent samples with Ad.CTP1-nLacZ and Ad.CTP3-nLacZ viruses: in bothcases exposure of liver and tumour tissue resulted in a level ofexpression equivalent to CMV but restricted to tumour tissue. To date,all 5 colorectal metastases examined were permissive for high-level CTP1and CTP3 expression. Of 10 primaries, 3 were found to be weakly ornon-permissive for promoter activity. Of note, the primary tumours thatgave rise to the secondaries were permissive in each case.

Example 16

[0188] High-Level CTP-Mediated Expression Correlates with High Level,Non-Membranous Expression of Beta-Catenin

[0189] In Example 15 we demonstrate that CTP1 and CTP3 can providehigh-level gene expression selectively in secondary colorectal cancertissue despite the simultaneous introduction of the transgene intoneighbouring healthy liver tissue. Whilst to date the promoters wereactive in all secondary CRC deposits, low-level or undetectableexpression was observed in 3 of 10 primaries. Analysis of thesenon-permissive tumours for beta-catenin revealed a correlation betweenthe overall level and sub-cellular distribution of the protein. FIG. 18shows the results for representative permissive and non-permissivetumour samples. Tumour A (non-permissive) is relativelywell-differentiated with beta-catenin staining restricted largely to thecell periphery, consistent with this being associated with E-cadherin.Tumour B (permissive) in contrast is poorly differentiated with asignificantly higher level of cytoplasmic/nuclear beta-catenin staining.This finding provides further evidence for a dependence of the CTPpromoters on beta-catenin deregulation. It conflicts with the simplemodel of colon carcinogenesis in which beta-catenin deregulationresulting in constitutive activation of genes responsive tobeta-catenin/Tcf is the initiating event. A practical application ofthis finding is that it may provide the basis for pre-selection ofpatients possessing tumours that are permissive for the CTP promotersand thus potentially treatable by a gene therapy approach in which atherapeutic gene is under the control of a CTP promoter.

[0190] All references cited herein are hereby incorporated by referencein their entireties.

1 27 1 64 DNA Artificial Sequence misc_feature Synthetic Oligonucleotide1 ctagcaagct tactagtcct ttgatcaaga gtcctacctt tgatctctaa atgcaccttt 60gatc 64 2 72 DNA Artificial Sequence misc_feature SyntheticOligonucleotide 2 actgaattcc ttgatcaaag gtgcatttag agatcaaagg taggactcttgatcaaagga 60 ctagtaagct tg 72 3 60 DNA Artificial Sequence misc_featureSynthetic Oligonucleotide 3 aaggaattca gtcctttgat caagagtcct acctttgatctctaaatgca cctttgatca 60 4 52 DNA Artificial Sequence misc_featureSynthetic Oligonucleotide 4 gatctgatca aaggtgcatt tagagatcaa aggtaggactcttgatcaaa gg 52 5 128 DNA Artificial Sequence misc_feature SyntheticOligonucleotide 5 ctagcaagct tactagtcct ttgatcaaga gtttcctacc tttgatctctaaattgcacc 60 tttgatcaag gaattcagtc ctttgatcaa gagtaaccta cctttgatctctaaatgcac 120 ctttatca 128 6 128 DNA Artificial Sequence misc_featureSynthetic Oligonucleotide 6 gatctgataa aggtgcattt agagatcaaa ggtaggttactcttgatcaa aggactgaat 60 tccttgatca aaggtgcaat ttagagatca aaggtaggaaactcttgatc aaaggactag 120 taagcttg 128 7 107 DNA Artificial Sequencemisc_feature Synthetic Oligonucleotide 7 ctagcaagct tactagtcctttgatcaagc tacctttgat ctctagcacc tttgatcaag 60 agtcctttga tcaagcctacctttgatctc taaatgcacc tttatca 107 8 107 DNA Artificial Sequencemisc_feature Synthetic Oligonucleotide 8 gatctgataa aggtgcatttagagatcaaa ggtaggcttg atcaaaggac tcttgatcaa 60 aggtgctaga gatcaaaggtagcttgatca aaggactagt aagcttg 107 9 95 DNA Artificial Sequencemisc_feature Synthetic Oligonucleotide 9 ctagcaagct tactagtcctttgatcaata cctttgatct cacctttgat caagtccttt 60 gatcatacct ttgatctctaaatgcacctt tatca 95 10 95 DNA Artificial Sequence misc_feature SyntheticOligonucleotide 10 gatctgataa aggtgcattt agagatcaaa ggtatgatcaaaggacttga tcaaaggtga 60 gatcaaaggt attgatcaaa ggactagtaa gcttg 95 11 16DNA Adenovirus type 5 11 gggtatataa tgcgcc 16 12 30 DNA ArtificialSequence misc_feature Synthetic Oligonucleotide 12 gaagatctcc ccttcttttctatggttaag 30 13 29 DNA Artificial Sequence misc_feature SyntheticOligonucleotide 13 gaagatctgc aatcattcgt ctgtttccc 29 14 30 DNAArtificial Sequence misc_feature Synthetic Oligonucleotide 14 gaagatctgatttggtcaat atgtgtacac 30 15 35 PRT Artificial Sequence misc_featureSynthetic Oligonucleotide 15 Lys Lys Lys Lys Lys Lys Gly Gly Phe Leu GlyPhe Trp Arg Gly Glu 1 5 10 15 Asn Gly Arg Lys Thr Arg Ser Ala Tyr GluArg Met Cys Asn Ile Leu 20 25 30 Lys Gly Lys 35 16 14 DNA ArtificialSequence misc_feature Synthetic Oligonucleotide 16 tacatctaga taat 14 1713 DNA Artificial Sequence misc_feature Synthetic Oligonucleotide 17ttatctagat gta 13 18 31 DNA Artificial Sequence misc_feature SyntheticOligonucleotide 18 ctagtatcga ttgttaatta agggcgtggc c 31 19 31 DNAArtificial Sequence misc_feature Synthetic Oligonucleotide 19 ttaaggccacgcccttaatt aacaatcgat a 31 20 167 DNA Artificial Sequence misc_featureSynthetic Oligonucleotide 20 ttgagatgca gatcgcagat ctgataaagg tgcatttagagatcaaaggt aggactcttg 60 atcaaaggac tgaattcctt gatcaaaggt gcatttagagatcaaaggta ggactctttg 120 atcaaaggga ctagtaagct tgctagcacg cgtaagagctcggtacc 167 21 138 DNA Artificial Sequence misc_feature SyntheticOligonucleotide 21 ggatgccaag ctttttagct tccttagctc ctgaaaatctcgccaagctg atgaattcga 60 gctggcgcat tatataccct ctagagtcga cggatcgagatctcgagccc gggctagcac 120 gcgtaagagc tcggtacc 138 22 131 DNA ArtificialSequence misc_feature Synthetic Oligonucleotide 22 atcgagatct gataaaggtgcatttagaga tcaaaggtag gttactcttt gaattcaggt 60 gcaatttaaa ggtaggaaactcttgatcaa aggactagta agcttgctag cacgcgtaag 120 agctcggtac c 131 23 125DNA Artificial Sequence misc_feature Synthetic Oligonucleotide 23atcgagatct gataaaggtg catttagaga tcaaaggtag gttactcttg atcaaaggac 60tgaattcagg aaactcttga tcaaaggact agtaagcttg ctagcacgcg taagagctcg 120gtacc 125 24 121 DNA Artificial Sequence misc_feature SyntheticOligonucleotide 24 atcgagatct gataaaggtg catttagaga tcaaaggtagtcacaggtgc aatttagaga 60 tcaaaggtag gaattgatca aaggatagta agcttgctagcacgcgtaag agctcggtac 120 c 121 25 132 DNA Artificial Sequencemisc_feature Synthetic Oligonucleotide 25 atcgagatct gataaaggttcttgatcaaa ggactgaatt ccttgatcaa aggtgcaatt 60 tagagatcaa aggtaggaaactcttgatca aaggactagt aagcttgcta gcacgcgtaa 120 gagctcggta cc 132 26 150DNA Artificial Sequence misc_feature Synthetic Oligonucleotide 26atcgagatcg ataaaggtgc atttagacga tcaaaggtag gttactcttg atcaaaggaa 60ttccttgatc aaaggtgcaa tttagagaag gtaggaaact cttgatcaaa ggactagtaa 120gcttgctagc acgcgtaaga gctcggtacc 150 27 244 DNA Artificial Sequencemisc_feature Synthetic Oligonucleotide 27 taccaacagt accggaatgccaagctagct ttttagcttc cttagctcct gaaaatctcg 60 ccaagctgat gaattcgagctggcgcatta tataccctct gataaaggtg catttagaga 120 tcaaaggtat gatcaaaggacttgatcaaa ggtgagatca aaggtattga tcaaaggact 180 agagcttact tagatcgcagatctcgagcc cgggctagca cgcgtaagag ctcggtacct 240 atcg 244

What is claimed is:
 1. A nucleic acid construct comprising: a T-cellfactor (TCF) response element comprising: at least one TCF bindingelement having the sequence CTTTGNN wherein N is A or T; an operablylinked promoter, and an expressible gene that is useful for thetreatment of a disease that is characterised by deregulation in Wntpathway signalling, wherein the expressible gene is operably linked toboth the TCF binding element and the promoter, which enables inducibleexpression of the gene.
 2. A nucleic acid construct comprising: a T-cellfactor (TCF) response element comprising: at least one TCF bindingelement having the sequence CTTTGNN wherein N is A or T; an operablylinked promoter, and an expressible gene that is useful for thetreatment of a disease that is characterised by the presence ofTCF/β-catenin heterodimers in diseased cells wherein the expressiblegene is operably linked to both the TCF binding element and thepromoter, which enables inducible expression of the gene.
 3. A nucleicacid construct comprising: a T-cell factor (TCF) response elementcomprising: at least one TCF binding element having the sequence CTTTGNNwherein N is A or T; an operably linked promoter, and an expressiblegene that is useful for the treatment of a cancer that is characterisedby the presence of TCF/β-catenin heterodimers in diseased cells whereinthe expressible gene is operably linked to both the TCF binding elementand the promoter, which enables inducible expression of the gene.
 4. Thenucleic acid construct of any one of claims 1 to 3 wherein theexpressible gene is selected from the group consisting of: a geneencoding a toxin, a prodrug-activating enzyme or an immunomodulatoryagent; a tumor-supressor gene or an apoptotic gene.
 5. The nucleic acidconstruct of claim 4, wherein the expressible gene encodes a toxin orprodrug-activating enzyme.
 6. The nucleic acid construct of claim 5,wherein the therapeutic gene encodes a nitroreductase capable ofactivating CB1954.
 7. The nucleic acid construct of any one of claims 1to 3 wherein the promoter is selected from the group consisting of theSV40 promoter, the E1B promoter, and the c-Fos promoter.
 8. The nucleicacid construct of claim 7, wherein the promoter is the E1B promoter. 9.A nucleic acid construct comprising: 1) a TCF response elementcomprising: at least 5 TCF binding elements having the sequence CTTTGNN,wherein N is A or T; and an operably linked promoter; and 2) anexpressible gene that is useful in the treatment of a disease that ischaracterised by the presence of TCF/β-catenin heterodimers in diseasedcells.
 10. The nucleic acid construct of claim 9 wherein the TCFresponse element comprises between 5 and 10 TCF binding elements. 11.The nucleic acid construct of claim 10 wherein the TCF response elementcomprises 5 TCF binding elements.
 12. A nucleic acid constructcomprising: 1) a TCF response element comprising: at least two TCFbinding elements having the sequence CTTTGNN, wherein N is A or T; andan operably linked promoter; and 2) an expressible gene that is usefulin the treatment of a disease that is characterised by the presence ofTCF/β-catenin heterodimers in diseased cells, wherein the expressiblegene is operably linked to the TCF response element which enablesinducible expression of the gene, and wherein the TCF binding elementsare separated from each other by between 3 and 20 nucleotides.
 13. Thenucleic acid construct of claim 12 wherein the TCF binding elements areseparated from each other by between 3 and 12 nucleotides.
 14. Thenucleic acid construct of claim 13 wherein the TCF binding elements areseparated from each other by between 10 and 12 nucleotides.
 15. Anucleic acid construct comprising: 1) a TCF response element comprising:at least one TCF binding element having the sequence CTTTGNN, wherein Nis A or T; and an operably linked promoter; and 2) an expressible genethat is useful in the treatment of a disease that is characterised bythe presence of TCF/1-catenin heterodimers in diseased cells, whereinthe expressible gene is operably linked to the TCF response elementwhich enables inducible expression of the gene, and wherein the TCFbinding element closest to the promoter is between 140 and 10nucleotides from the TATA box of the promoter.
 16. The nucleic acidconstruct of claim 15 wherein the promoter contains a TATA box, and theTCF binding element closest to the promoter is between 100 and 10nucleotides from the TATA box of the promoter.
 17. The nucleic acidconstruct of claim 16 wherein the promoter contains a TATA box, and theTCF binding element closest to the promoter is between 50 and 10nucleotides from the TATA box of the promoter.
 18. The nucleic acidconstruct of claim 17 wherein the promoter contains a TATA box, and theTCF binding element closest to the promoter is between 30 and 15nucleotides from the TATA box of the promoter.
 19. The nucleic acidconstruct of claim 12 wherein the TCF binding elements are separatedfrom each other by 3 or 4 nucleotides and wherein the promoter comprisesa TATA box, and the TCF binding element closest to the promoter is 25nucleotides from the TATA box of the promoter.
 20. The nucleic acidconstruct of any one of claims 1 to 3, 9, 12, 15, 29, 30 or 31, whereinthe TCF binding element has the nucleotide sequence CTTTGAT.
 21. Avector comprising the nucleic acid construct of any one of claims 1 to3, 9, 12, 15, 29, 30 or
 31. 22. A host cell transfected with the vectorof claim
 21. 23. A method of treatment of a disease characterised by aderegulation in Wnt pathway signalling or the presence of TCF/β-cateninheterodimers in diseased cells, comprising administering to a patient inneed of such treatment an effective dose of the nucleic acid constructof any one of claims 1 to 3, 9, 12 or
 15. 24. A method of treatment of adisease characterised by a deregulation in Wnt pathway signalling or thepresence of TCF/β-catenin heterodimers in diseased cells, comprisingadministering to a patient in need of such treatment an effective doseof the vector of claim
 21. 25. A method of treatment of a diseasecharacterised by a deregulation in Wnt pathway signalling or thepresence of TCF/β-catenin heterodimers in diseased cells, comprisingadministering to a patient in need of such treatment an effective doseof the host cell of claim
 22. 26. A composition comprising the nucleicacid construct of any of claims 1 to 3, 9, 12 or 15 and apharmaceutically acceptable excipient.
 27. A composition comprising thevector of claim
 21. 28. A composition comprising the host cell of claim22 and a pharmaceutically acceptable excipient.
 29. The nucleic acidconstruct according to claim 9, wherein the expressible gene is selectedfrom the groups consisting of: a gene encoding a toxin, a prodrugactivating enzyme, or an immunomodulatory element; a tumor suppressorgene; and an apoptotic gene.
 30. The nucleic acid construct according toclaim 12, wherein the expressible gene is selected from the groupsconsisting of: a gene encoding a toxin, a prodrug activating enzyme, oran immunomodulatory element; a tumor suppressor gene; and an apoptoticgene.
 31. The nucleic acid construct according to claim 15, wherein theexpressible gene is selected from the groups consisting of: a geneencoding a toxin, a prodrug activating enzyme, or an immunomodulatoryelement; a tumor suppressor gene; and an apoptotic gene.